The infectious agent of scrapie and other transmissible spongiform encephalopathies (TSE) resembles a virus in that it replicates in vivo and has distinct strains. Nevertheless, the agent has been proposed to contain only PrP-res, a neuropathogenic, protease-resistant form of the host-encoded prion protein. We have shown previously that PrP-res is derived post-translationally from normal, protease-sensitive PrP (PrP- sen) in scrapie-infected cells. However, the conversion mechanism and the relationship of PrP-res formation to TSE agent replication remain unclear, in part because the conversion was never accomplished in a defined, cell-free system. We have now converted PrP-sen to protease- resistant forms similar to scrapie associated PrP-res in a cell-free system composed of substantially purified constituents. This conversion required the presence of preexisting PrP-res, providing the first direct evidence that PrP-res derives from interactions between PrP-sen and PrP- res. Denaturation of the preexisting PrP-res prevented conversion, indicating that maintenance of some native structure of PrP-res was required. Our present studies with this reconstitution system are aimed at defining the conversion mechanism and specificity of the conversion as it relates to PrP genotype and scrapie strain. With further refinements of this system, a clear evaluation of how protease-resistant PrP relates to TSE infectivity may finally be possible. Another approach we have taken to defining the mechanism of PrP-res formation and how it might be blocked for therapeutic purposes is to identify and characterize inhibitors of PrP-res accumulation scrapie- infected mouse neuroblastoma cells. Having already shown that Congo red and certain sulfated glycans are potent inhibitors, we proceeded to compare the potencies of numerous additional sulfated and sulfonated polymers and Congo red analogs in order to better define the attributes of such inhibitors. These studies have shown that there is great molecular specificity in the inhibition mechanism because even subtle changes in inhibitor structure, such as moving a pair of methyl groups on the biphenyl group of Congo red analogs, can cause dramatic differences in potency. Such specificity is important in potential pharmacological applications of such inhibitors.